This project uses a combination of novel single-molecule and bulk biochemical methods to study non- homologous end joining (NHEJ), the predominant mechanism of DNA double strand break repair in human cells. During NHEJ, DNA ends are first recognized and bound by the Ku70/80 heterodimer, which, in turn, recruits a number of other factors that are responsible for bridging the ends, processing them for ligation and ultimately covalently joining them. It remains unclear how these NHEJ factors tether DNA ends in a synaptic complex and how end synapsis, processing, and ligation are coordinated. We will use single-molecule FRET approaches to observe NHEJ in real time in Xenopus egg extracts. These assays will allow us to characterize previously undetectable intermediate steps in the NHEJ mechanism. The completion of the following aims will give us increased molecular insight into the repair of double strand breaks, such as those induced by radiation therapy or exploited by emerging genome editing technologies. Aim 1) Determine how DNA ends are tethered during non-homologous end joining Broken DNA ends must be tethered efficiently to prevent them from diffusing apart and joining to the wrong partner. In vitro reconstitution experiments have implicated nearly every core NHEJ factor in end tethering, yet it remains unclear what role different factors play in the synapsis of DNA ends under physiological conditions. Using single-molecule FRET assays that we have developed in my lab, we will follow the synapsis of DNA ends in real time and characterize the NHEJ factors responsible for their tethering. Aim 2) Determine how the composition of the NHEJ machinery changes during end joining in this aim, we will determine the composition of different synaptic intermediates and investigate how DNA-PK activity regulates the composition of the synaptic complex. First, we will develop bulk biochemical methods to trap the distinct synaptic intermediates identified by our single-molecule experiments, and we will analyze their composition by mass spectrometry. Next, we will carry out single-molecule imaging experiments to characterize the association of XRCC4-LIG4 and XLF with different synaptic complexes and determine how this association is regulated by DNA-PK activity. Aim 3) Elucidate how end processing is regulated during NHEJ to maintain fidelity DNA ends that are chemically damaged or otherwise incompatible must be processed to permit ligation. It remains unclear how the NHEJ machinery regulates processing so as to minimize mutations. One possibility, supported by recent deep-sequencing of NHEJ junctions, is that ligation is attempted within the synaptic complex prior to any processing. In ths aim, we will test whether factors that are required for synaptic complex formation are also required to initiate processing at different types of DNA ends and use single-molecule imaging to directly visualize the processing and attempted ligation of substrates with incompatible ends.